Varying an illumination path of a selective plane illumination microscopy

ABSTRACT

A system for illuminating a microscopy specimen includes an illumination source configured to emit a light that travels along an illumination path to illuminate the microscopy specimen placed on an optical detection path of an optical microscope. The system also includes optical elements in the illumination path and configured to at least in part transform the light from the illumination source into a light sheet illuminating the microscopy specimen. The optical elements include an electronically tunable lens configured to vary a focal distance of the electronically tunable lens to dynamically vary a position of a waist of the light sheet illuminating the microscopy specimen. The optical elements include a deflector configured to vertically move the light sheet to illuminate the microscopy specimen at different horizontal planes.

CROSS REFERENCE TO OTHER APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/944,485, entitled VARYING AN ILLUMINATION PATH OF A SELECTIVE PLANEILLUMINATION MICROSCOPY filed Apr. 3, 2018 which is incorporated hereinby reference for all purposes, which is a continuation in part of U.S.patent application Ser. No. 15/680,075 entitled EXTENDING OPTICALMICROSCOPES TO PROVIDE SELECTIVE PLANE ILLUMINATION MICROSCOPY filedAug. 17, 2017, now U.S. Pat. No. 10,365,464, which is incorporatedherein by reference for all purposes, which claims priority to U.S.Provisional Patent Application No. 62/489,168 entitled EXTENDING OPTICALMICROSCOPES TO PROVIDE SELECTIVE PLANE ILLUMINATION MICROSCOPY filedApr. 24, 2017 which is incorporated herein by reference for allpurposes.

U.S. application Ser. No. 15/944,485 claims priority to U.S. ProvisionalPatent Application No. 62/556,093 entitled OPTICAL ARRANGEMENT TO EXTENDOPTICAL MICROSCOPES TO PROVIDE 3D SELECTIVE PLANE ILLUMINATIONMICROSCOPY filed Sep. 8, 2017 which is incorporated herein by referencefor all purposes.

BACKGROUND OF THE INVENTION

Light sheet fluorescence microscopy or selective plane illuminationmicroscopy (SPIM) technology typically relies on illuminating of aspecimen in thin optical slices, formed from laser light, exciting thefluorophores in the specimen and acquiring light emitted by theilluminated plane inside the specimen. The direction in which the lightis detected is typically perpendicular to the illuminated plane. Theresolution of SPIM is often limited by the shape and properties of thelight sheet illuminating the specimen.

With SPIM, the lateral resolution is determined by the detectionobjective lens and the axial resolution is related to the numericalaperture (NA) of the illumination objective. With higher NAs, the axialresolution is similar to confocal fluorescence microscopes. Images fromlight sheet microscopes exhibit a better signal-to-noise (S/N) ratio anda higher dynamic range than images produced by confocal fluorescencemicroscopes. With low NAs, the axial resolution is determined by thethickness of the light sheet at its thinnest point (i.e., the waist),with an excellent isotropic Point Spread Function. However, while theaxial resolution increases only linearly with increasing NA, the fieldof view (i.e., FOV) with optimal axial resolution decreases with thesquare of the NA. This relation results in a fundamental design problem,where a large FOV is not compatible with high axial resolution.Therefore, there is a need for an illumination system that produces thethinnest possible light sheet illumination over the largest possiblefield of view.

BRIEF DESCRIPTION OF THE DRAWINGS

Various embodiments of the invention are disclosed in the followingdetailed description and the accompanying drawings.

FIG. 1A is a diagram illustrating an embodiment of components of asystem that can be coupled to a microscope to convert the microscope toperform SPIM.

FIG. 1B is a diagram illustrating an embodiment showing an add-on systemadded to a vertical microscope to convert the vertical microscope toperform SPIM.

FIG. 2 is a diagram illustrating an example of illumination of aspecimen.

FIG. 3A-FIG. 3D are diagrams illustrating different views of a firstembodiment of an illumination path and optical components of anillumination unit.

FIG. 4A-FIG. 4D are diagrams illustrating different views of a secondembodiment of an illumination path and optical components of anillumination unit.

FIG. 5A-FIG. 5D are diagrams illustrating different views of a thirdembodiment of an illumination path and optical components of anillumination unit.

FIG. 6A-FIG. 6E are diagrams illustrating various embodiments offocusing unit add-ons to an optical microscope.

FIG. 7 is a diagram illustrating an embodiment of a specimen holdingchamber assembly.

FIG. 8 is a diagram illustrating an embodiment of a mold-formed specimenholder over a T-spike rotary mounting.

FIG. 9 is a flowchart illustrating an embodiment of a process forforming a molded specimen holder.

FIGS. 10A-10H illustrate an embodiment of various steps of forming amolded specimen holder.

DETAILED DESCRIPTION

The invention can be implemented in numerous ways, including as aprocess; an apparatus; a system; a composition of matter; a computerprogram product embodied on a computer readable storage medium; and/or aprocessor, such as a processor configured to execute instructions storedon and/or provided by a memory coupled to the processor. In thisspecification, these implementations, or any other form that theinvention may take, may be referred to as techniques. In general, theorder of the steps of disclosed processes may be altered within thescope of the invention. Unless stated otherwise, a component such as aprocessor or a memory described as being configured to perform a taskmay be implemented as a general component that is temporarily configuredto perform the task at a given time or a specific component that ismanufactured to perform the task. As used herein, the term ‘processor’refers to one or more devices, circuits, and/or processing coresconfigured to process data, such as computer program instructions.

A detailed description of one or more embodiments of the invention isprovided below along with accompanying figures that illustrate theprinciples of the invention. The invention is described in connectionwith such embodiments, but the invention is not limited to anyembodiment. The scope of the invention is limited only by the claims andthe invention encompasses numerous alternatives, modifications andequivalents. Numerous specific details are set forth in the followingdescription in order to provide a thorough understanding of theinvention. These details are provided for the purpose of example and theinvention may be practiced according to the claims without some or allof these specific details. For the purpose of clarity, technicalmaterial that is known in the technical fields related to the inventionhas not been described in detail so that the invention is notunnecessarily obscured.

High spatial and temporal resolution for three-dimensional light sheetimaging is critical for the understanding of physiological processes ofliving specimens while keeping them in their natural state withoutperturbation. The typical methods based on mechanical motion control forvolume acquisition introduce vibrations during the acquisition and limitthe scanning speed. As water-dipping objectives are customarily used forobservation of biologic specimens in the specimens' natural medium,perturbations from a moving detection objective may influence thespecimen behavior under observation and restrain the scope ofapplication for dynamic studies. Larger specimens can also exceed thelaser waist (focus) area and reduce the optical sectioning power of thelight sheet assembly.

Optical arrangements (e.g., serving as add-on attachments to variousinterfaces of an optical microscope) providing illumination for athree-dimensional selective plane light-sheet microscopy (SPIM) aredisclosed. In some embodiments, these optical arrangements candynamically vary: a cross section of a light sheet, a position of awaist of the light sheet along an axis of illumination, a position ofthe plane of the light sheet illumination, and/or a direction in whichbeam components extending within the light sheet are directed to thespecimen. Additionally, a focus distance of a detection lens in anoptical detection path of a microscope can also be dynamically and/orautomatically varied and synchronized with the dynamic variance of thelight sheet illumination to increase the resolution of a detected imageof the SPIM specimen.

Typical SPIM solutions are offered as stand-alone digital systems with afar different operating approach than conventional optical microscopy.Using traditional SPIM microscopes requires special training and imposesnew behaviors upon the users, thus minimizing their productivity andlimiting the market penetration and scientific community's awareness ofadvantages offered by the light sheet technology. In some embodiments, avertical optical microscope (e.g., upright or inverted microscope) isconverted to provide selective plane illumination microscopy. Forexample, by adding components to a traditional vertical opticalmicroscope to convert it to provide selective plane illuminationmicroscopy, cost savings and reduced physical size footprint areachieved as compared to using a traditional dedicated standalone SPIMmicroscope. For example, a typical lab setting already includes atraditional vertical optical microscope and allowing the traditionalvertical optical microscope to be converted to an SPIM microscope savescosts and space. An illumination source is configured to generate alight sheet along a longitudinal axis to illuminate a specimen placed ina vertical optical detection axis of the vertical optical microscope.The illumination source is configured to generate a light sheet along alongitudinal axis that is substantially perpendicular to a verticaloptical detection axis of the vertical optical microscope and theillumination source is configured to produce an excitation at a plane inthe specimen that generates fluorescent emissions. A detection sensor isplaced in the detection optical path of the vertical optical detectionaxis of the vertical optical microscope. The detection sensor isconfigured to detect the fluorescent emissions to provide selectiveplane illumination microscopy.

Typical standalone SPIM microscopes are configured in a horizontalorientation. For example, both the illumination path and the detectionpath are oriented horizontally (e.g., in the horizontal planesubstantially perpendicular to the direction of gravity). For example,typical solutions include an excitation illumination source objectivehaving the excitation illumination axis and the detection objectivehaving the detection optical axis that are both engaged to the samemount body, where the two axes are oriented in a perpendicular relationto each other in the horizontal plane. This often is due to limitationsin traditional specimen holding solutions. For example, SPIM is oftenutilized to observe biological specimens suspended in a fluid andlimitations of how the specimen can be contained and rotated usingtraditional specimen holding solutions require the specimen to beilluminated and detected in the horizontal plane. However, the detectionoptical path of traditional vertical optical microscopes is in thevertical direction. Solutions described herein allow SPIM detection tobe achieved using the vertical optical microscope's optical arrangementin the vertical direction.

In some embodiments, both observation and acquisition modes are added tothe microscope detection objective's optical arrangement of verticaloptical microscopes. By using the microscope stand of the verticaloptical microscope as an integral part of the detection unit, it takesadvantage of quality optical elements already present in the detectionpath (e.g., including objective turret, filter wheel, binoculars, andvideo port), thereby reducing complexity of building a selective planeillumination microscopy system. As no alterations to the detectionpath's optics of the vertical optical microscope are introduced, allother functionalities that could be necessary for other observationmodes (e.g., transmission, wide field fluorescence, etc.) are keptunaltered, including convenient means for specific applications such aselectrophysiology. Therefore it serves as an upgrade on existingmicroscopy platforms by adding light sheet imaging capabilitiesproviding a cost effective solution or as a whole system by integratinga functional fluorescence microscope.

FIG. 1A is a diagram illustrating an embodiment of components of asystem that can be coupled to a microscope to convert the microscope toperform SPIM. System 100 includes illumination units 102 and 104, andstepper stage 106.

Illumination units 102 and 104 are designed to work with a laser source(e.g., fiber laser source) to produce a light sheet using a cylindricallens. This allows direct imaging of an optical section with a singleframe at full camera resolution. For better illumination planehomogeneity across the specimen, two illumination units are used on bothsides of a specimen to compensate the absorption effects with a thickspecimen. In some embodiments, the light sheet is projected using anobjective, which can be adapted according to specimen size and detectionmagnification. The illumination units are designed to compensatechromatic shift for the visible spectrum, thus allowing the simultaneousillumination at several wavelengths using a laser combiner formulti-fluorescence imaging. Although two illumination units are shown, asingle or any other number of illumination units may be utilized invarious other embodiments. In some embodiments, illumination units 102and/or 104 produce a pencil beam rather than or in addition to a lightsheet.

Optical arrangements of illumination units 102 and/or 104 provideillumination for a three-dimensional selective plane light-sheetmicroscopy. In some embodiments, these optical arrangements candynamically vary: a cross section of a light sheet, a position of awaist of the light sheet along an axis of illumination, a position ofthe plane of the light sheet illumination, and/or a direction in whichbeam components extending within the light sheet are directed to thespecimen.

Stepper stage 106 includes a motorized translation stage to move thespecimen through the illumination plane of illumination units 102 and104. Thus, using stepper stage 106, the illumination sheet and thedetection plane may remain in fixed positions while detecting variousslices as the translation stage is moved in steps. The shown stepperstage 106 includes a support for a specimen chamber, a z-stage that ismoveable in the vertical z-direction via a motor, a slider, and controlsfor x and y position adjustments of the stage in the horizontal plane.In some embodiments, a base configured to engage a specimen stage forsupporting and orienting the specimen holder in an x-y direction isutilized. In some embodiments, a translational stage configured toengage the specimen holder in the z-direction is utilized.

FIG. 1B is a diagram illustrating an embodiment showing an add-on systemadded to a vertical microscope to convert the vertical microscope toperform SPIM. For example, system 100 of FIG. 1A is shown engaged withvertical optical microscope 110. Vertical optical microscope 110 shownin this example is a trinocular fluorescence microscope equipped with afilter wheel and an objective turret with a water dipping/immersiondetection lens. However in various other embodiments, other types ofoptical microscopes may be utilized. The optical detection path utilizedto perform SPIM may utilize standard components of microscope 110,including its components in the optical detection path (e.g., objectivelenses, arm, filter in filter wheel, etc.). An output port of opticalmicroscope 110 is coupled to detection unit 112 that is utilized toacquire the SPIM image detected using the optical detection path ofmicroscope 110. For example, detection unit 112 includes a digitalcamera. In some embodiments, a focus distance of the optical detectionpath can be dynamically varied and synchronized with the dynamicvariance of the light sheet illumination to increase the resolution of adetected image of the SPIM specimen.

Specimen chamber and holder assembly 116 has been configured to handleSPIM using a vertical optical detection path configuration as comparedto traditional holders that have been designed to be utilized forhorizontal optical SPIM detection paths. Specimen chamber and holderassembly 116 allows a specimen to be rotated about a substantiallyhorizontal rotational axis and substantially perpendicular to theoptical axis of the detection objective using a rotational drive orknob. For example, specimen chamber and holder assembly 116 embeds aspecimen in a substantially rigid substantially transparent embeddingcompound maintained in an immersion liquid and placed in the holder,allowing the specimen to be rotated about the substantially horizontalrotational axis that is substantially perpendicular to the optical axisof the detection objective. In some embodiments, specimen chamber andholder assembly 116 is supported by a specimen stage for supporting andorienting assembly 116 in an x-y direction and/or a translational stageconfigured to engage the assembly 116 in the z-direction.

FIG. 2 is a diagram illustrating an example of illumination of aspecimen. In some embodiments, a light-sheet microscopy system uses astandard upright or inverted microscope, capable of illuminating a setof planes within a specimen, to detect the fluorescent emission comingfrom the illuminated plane, while at the same time producing the finestaxial resolution at the largest region of interest. Specimen 200 isbeing illuminated by illumination objective 202 and illuminationobjective 204. The illuminated specimen is observed via opticaldetection objective 206. In some embodiments, detection objective 206 isa part of microscope 110, illumination objective 202 is a part ofillumination unit 102, and illumination objective 204 is a part ofillumination unit 104 of system 100 of FIGS. 1A and 1B. For example, theillumination units are designed to work with fibered laser sources toproduce a light sheet using cylindrical lenses of the objectives. Thisallows direct imaging of an optical section with a single frame at fullcamera resolution. For better illumination plane homogeneity across thespecimen, two illumination units are used on both sides of the specimento compensate for the absorption effects of a thick specimen sample. Inan alternative embodiment, a single illumination unit is utilized. Insome embodiments, the light sheet is projected using finite-infiniteobjectives, which can be adapted according to specimen size anddetection magnification. In some embodiments, the illumination output ofthe objectives has a cross section of an elongated ellipse due to anassembly of optical elements in which the thin sheet of light isgenerated from one or many laser light sources. In another embodiment,the illumination output of the objectives has a cross section of anelongated rectangle. Lenses of objectives 202 and 204 are designed tooptically compensate chromatic shift for the visible spectrum, thusallowing the simultaneous illumination at several wavelengths using alaser combiner for multi-fluorescence imaging.

Illumination objectives 202 and 204 focus the laser light source tocreate a light sheet. However, as shown in FIG. 2, due to the focusingof the light source by the lens of the objective, the light sheet isthinner at the focal point area (i.e., at the “waist”) and becomesthicker away from the focal point area. A thinner light sheet allows forbetter image resolution and thus a uniformly thin light sheet isdesired. Given the effects of the shown divergence, a light sheet withina limited range of thickness can be utilized in order to maintain adesired image resolution, thus limiting the field of view to the area ofthe light sheet within the thickness limit. However in some cases it maybe desirable to capture a specimen that is larger than the limited fieldof view. In some embodiments, a variable focus lens is utilized inillumination units to sweep the focal point across the specimen tocreate a thinner light sheet over a larger area of the light sheet. Forexample, focus distance of the illumination is automatically adjusted tomove and sweep the focus across the specimen during image capture toautomatically sweep the thinnest point of the light sheet across thespecimen. By using a lens of variable focal distance in the illuminationpath of the optical arrangement, sweeping of the light sheet waist ismade possible along the illumination direction. The lens of variablefocal distance can vary between both negative and positive opticalpowers. This allows the acquisition of light sheet images of both thefinest axial resolution and the largest field of view in one and thesame frame.

By synchronizing lines of a rolling shutter of a detector (e.g., digitalcamera) with the sweeping position of the waist of the light sheet, adetected image of the specimen with a larger field of view can begenerated without physically moving the specimen within the plane of thelight sheet. The focus distance of the illumination may be adjustedelectronically and/or mechanically. For example, rather than relying onthe mechanical motor mechanism that may introduce vibrations, the focaldistance of an electronically tunable lens may be changed electrically(e.g., via electromagnets, piezoelectric element, current through asolution, etc.) without a use of a motor.

In some embodiments, a lens of variable focal distance is utilized inthe detection path of the microscope. This optical device, allowing forfast remote focusing, is inserted into the detection path between themicroscope's video output port and the digital camera, or between themicroscope's detection objective and the microscope's tube lens. Thespecimen, in its chamber, is set in a fixed position when theillumination plane and the detection plane move simultaneously throughthe specimen. As the specimen remains in a steady position, vibrationsand perturbation issues are alleviated. Incidentally specimen mountingand holding becomes much easier. Three dimensional acquisition can beachieved at camera frame rate without being limited by mechanicalconstraints (e.g., 100 fps at 4Mpixels using a sensitive sCMOS camera).

In some embodiments, if required, a means for influencing the lightsheet direction is utilized. Together with the sweeping of the lightsheet waist, this means helps reduce or remove altogether shadowsoccurring within the observed specimen's plane. By integrating thesescanning means, the light sheet system not only provides opticalsectioning with optimal spatial resolution and signal to noise ratio,but also delivers unprecedented temporal resolution for 3D acquisition,addressing the needs for dynamic imaging of rapid biophysical processes.

FIG. 3A-FIG. 3D are diagrams illustrating different views of a firstembodiment of an illumination path and optical components of anillumination unit. For example, the shown optical components areincluded and arranged in the shown relative order in illuminationcomponent 102 of FIGS. 1A-1B. In some embodiments, two illuminationunits are utilized and the shown optical components are included andarranged in the corresponding relative order (e.g., mirrored on z-planefrom shown arrangement) in illumination component 104 of FIGS. 1A-1B.Not all components of the illumination have been shown. FIG. 3A shows aprofile view. FIG. 3B shows a front view. FIG. 3C shows a top view. FIG.3D shows a side view. The Z-direction axis is the vertical axis.

Illumination path 300 shows a path of light from light source 302 to aspecimen in specimen chamber 318 as the light is manipulated by opticalcomponents to produce a light sheet to illuminate the specimen. Anexample of light source 302 is a laser light source that produces alaser light (e.g., laser beam). The light (e.g., light bundle ofcoherent light) from laser source 302 passes through collimator 304 thataligns the beams of the light using one or more optical lenses.Collimator 304 includes and/or is followed by one or more componentswith horizontal and vertical slits with adjustable opening widths thatallow adjustment of the aperture and field stops. The adjustment of thevertical opening width implements the field stop that influences thewidth of the light sheet, while the adjustment of the horizontal openingimplements the aperture stop that influences the length of the waist(e.g., Length=2×sqrt(2)×Rayleigh length) and the height of the waist ofthe light sheet. One or more utilized diaphragms (e.g., included in ordownstream of the collimator and realizing field and/or angular aperturestops for the light sheet) can be arranged on a changer wheel ormaintained in place. The widths of apertures of the diaphragms may beset manually or automatically. A cylindrical lens, for example, can beused as an aspherical optical element.

Next, the light following illumination path 300 is deflected byhorizontal optical deflection component 306. An example of horizontaloptical deflection component 306 is an oscillating mirror. When opticaldeflection component 306 is oscillated, it generates scanning horizontalmovements of the light sheet (e.g., to reduce shadows in illumination ofthe specimen). The oscillation of horizontal optical deflectioncomponent 306 includes a back and forth rotation (e.g., vibration) abouta central rotational axis (e.g., on the x-axis). This oscillation may beachieved via a piezoelectric, mechanical, and/or other electromechanicalcomponent. Horizontal optical deflection component 306 is positioneddownstream of collimator 304 and diaphragms realizing field and angularaperture stops for the light sheet. As the result of oscillatingmovement of horizontal optical deflection component 306, the light beamcomponents of the light sheet strike the specimen in specimen chamber318 at alternating directions many times so as to reduce or removealtogether the shadows caused by opaque specimen substances within theilluminated light sheet plane that might appear in the path of anyindividual beam component. Thus horizontal optical deflection component306 enables scanning automatic movement of the horizontal back and forthposition (e.g., y-axis direction) of the waist of the light sheetilluminating the specimen.

Then the light following illumination path 300 passes through asphericalcomponent 308 that includes one or more aspherical (e.g., cylindricallens) optical lens elements (e.g., generates a light bundle with anelliptic cross section).

Then the light following illumination path 300 passes through variablefocus lens component 310. An example of variable focus lens component310 includes an electronically tunable lens with an electronicallyvariable focal distance (e.g., changes focus via electromagnets,piezoelectric element, current through a solution, etc.). Variable focuslens component 310 can be utilized to sweep the focal point across thespecimen to create a thinner light sheet over a larger area of the lightsheet. For example, focal distance of the illumination lens isautomatically and/or continually adjusted/swept across a range of focaldistance values to move the focus of the lens across the width of thespecimen during image capture to sweep the thinnest point of the lightsheet across the specimen. The focal distance of variable focus lenscomponent 310 can be dynamically and automatically tuned/scanned tochange the diopter of variable focus lens component 310 in a range thatincludes both positive and negative diopter values (e.g., between bothnegative and positive optical powers). For example, variable focus lenscomponent 310 can act both as a converging and diverging lens byelectronically adjusting a parameter of variable focus lens component310. By introducing a lens of variable focal distance into theillumination path of the optical arrangement, sweeping of the lightsheet waist is made possible along the illumination direction. Thusvariable focus lens component 310 enables automatic movement (e.g.,scan) of the horizontal side to side position (e.g., x-axis direction)of the waist of the light sheet illuminating the specimen. In someembodiments, variable focus lens component 310 is positioned at or closeto planes optically conjugated with an aperture of illuminationobjective 316.

Then the light following illumination path 300 is deflected by avertical optical deflection component 312. An example of verticaloptical deflection component 312 is an oscillating mirror. Theoscillation of vertical optical deflection component 312 includes a backand forth rotation (e.g., vibration) about a central rotational axis(e.g., on the y-axis). This oscillation may be achieved via apiezoelectric, mechanical, and/or other electromechanical component.When optical deflection component 312 is oscillated, it generatesscanning vertical movements of the light sheet. For example, to achievea scanning vertical (e.g., perpendicular to the plane of the lightsheet) movement of the light sheet plane, vertical optical deflectioncomponent 312 is positioned downstream of aspherical component 308generating a light bundle with an elliptic cross section. As the resultof linear or oscillating movement of vertical optical deflectioncomponent 312, the light beam components of the light sheet strike thespecimen at a series of planes thus achieving optical slicing of thespecimen, allowing collection of fluorescent emission emitted inconsecutive slices within the specimen, by a detector (e.g., digitalcamera), or to observe the slices in real time directly with anobservation lens arrangement (e.g., binoculars). Thus vertical opticaldeflection component 312 enables automatic movement (e.g., scan) of thevertical position (e.g., z-axis direction) of the waist of the lightsheet illuminating the specimen.

Then the light following illumination path 300 passes through opticalrelay lens component 314. Optical relay lens component 314 includesoptical lenses and extends the illumination path and directs the lightbundle to the back aperture of illumination objective 316. Using one ormore optical lenses, illumination objective 316 emits and focuses thelight sheet on the specimen in specimen chamber 318, which is set toemit fluorescent light.

As the result of movement (e.g., linear, oscillating, or other) of thefocus of optical components with variable focusing distance, theposition of the waist of the light sheet is altered, which results inthe illuminating of the substances of the specimen with the light sheetof the thinnest cross section over the widest range along the axis ofillumination. This makes it possible to maintain the as fine as possibleaxial resolution of the light sheet microscopy (e.g., for detectionobjectives with low to medium numerical aperture (NA), it is determinedby the thickness of the light sheet waist) and/or discard the signalsfrom the neighboring slices of the specimen (e.g., for detectionobjectives with high NA), while imaging at as large as possible a regionof interest within the specimen, and, if required, to achieve reductionin shadows occurring within the observed specimen plane as a result ofthe light sheet illumination.

FIG. 4A-FIG. 4D are diagrams illustrating different views of a secondembodiment of an illumination path and optical components of anillumination unit. For example, the shown optical components areincluded and arranged in the shown relative order in illuminationcomponent 102 of FIGS. 1A-1B. In some embodiments, two illuminationunits are utilized and the shown optical components are included andarranged in the corresponding relative order (e.g., mirrored on z-planefrom shown arrangement) in illumination component 104 of FIGS. 1A-1B.Not all components of the illumination have been shown. FIG. 4A shows aprofile view. FIG. 4B shows a front view. FIG. 4C shows a top view. FIG.4D shows a side view. The z-direction axis is the vertical axis. Adifference between illumination path 400 of FIG. 4A-FIG. 4D andillumination path 300 of FIG. 3A-FIG. 3D includes a location of variablefocus lens component 310 in the illumination path relative to the otheroptical components. In illumination path 400, variable focus lenscomponent 310 is positioned after vertical optical deflection component312, in between vertical optical deflection component 312 and opticalrelay lens component 314. This different relative positioning ofvariable focus lens component 310 allows the shown components to fit ina different physical form factor that may be desired or necessary incertain configurations.

FIG. 5A-FIG. 5D are diagrams illustrating different views of a thirdembodiment of an illumination path and optical components of anillumination unit. For example, the shown optical components areincluded and arranged in the shown relative order in illuminationcomponent 102 of FIGS. 1A-1B. In some embodiments, two illuminationunits are utilized and the shown optical components are included andarranged in the corresponding relative order (e.g., mirrored on z-planefrom shown arrangement) in illumination component 104 of FIGS. 1A-1B.Not all components of the illumination have been shown. FIG. 5A shows aprofile view. FIG. 5B shows a front view. FIG. 5C shows a top view. FIG.5D shows a side view. The z-direction axis is the vertical axis. Adifference between illumination path 500 of FIG. 5A-FIG. 5D andillumination path 300 of FIG. 3A-FIG. 3D includes a location of variablefocus lens component 310 in the illumination path relative to the otheroptical components. In illumination path 500, variable focus lenscomponent 310 is positioned after optical relay lens component 314, inbetween optical relay lens component 314 and illumination objective 316.This different relative positioning of variable focus lens component 310allows the shown components to fit in a different physical form factorthat may be desired or necessary in certain configurations.

FIG. 6A-FIG. 6E are diagrams illustrating various embodiments offocusing unit add-ons to an optical microscope. High spatial andtemporal resolution for a 3D light sheet imaging allows observation ofphysiological processes of living specimens while keeping them in theirnatural state without perturbation. Typical methods based on mechanicalmotion of the specimen for volume acquisition introduce vibrationsduring the acquisition and limit the scanning speed. As water-dippingobjectives are customarily used for observation of biologic specimens inthe specimens' natural medium, perturbations from a moving detectionobjective may influence the specimen behavior under observation andrestrain the scope of application for dynamic studies. Larger specimenscan also exceed the laser waist (focus) area and reduce the opticalsectioning power of the light sheet assembly. In some embodiments, fastand vibration free 3D acquisition is performed using tunable lenses. Forexample, in order to generate a 3D image of a specimen using SPIM, eachslice of the specimen at different depths is illuminated using a lightsheet and captured using a lens focused on the slice. The image of thespecimen at different depths then can be digitally processed andcombined to generate a 3D rendering of the specimen. One way to capturethe various slices of the specimen at different depths is to physicallymove the specimen up and down and/or side to side in increments. Howeveras discussed previously, vibrations introduced in physically moving thespecimen may lead to drawbacks.

In some embodiments, instead of moving the specimen for scanning thedepth of the specimen, a scanning device based on the lens ofdynamically variable focal distance is utilized. Video output focusingunit 602 may be inserted into the detection path between a microscope'svideo output port and a digital camera, and detection objective outputfocusing unit 620 may be inserted between the microscope's detectionobjective and the microscope turret and/or tube lens. One or both offocusing unit 602 and focusing unit 620 may be utilized in variousembodiments. Inserting the focusing unit between the optical microscopeand the digital camera provides a possibility to switch between severalmicroscope objectives attached to the microscope turret. Inserting thefocusing unit between the optical objective and the objective turret ofthat microscope provides for wider vertical scanning range. Insertingtwo focusing units, each containing at least one lens of a variablefocal distance, is also possible. In that case, one lens (or a setthereof) of a variable focal distance serves as a means to select anaverage height of the light sheet plane within the specimen, while theother (or the set thereof) serves as a means of selecting a series ofplanes around the latter plane.

The specimen sample (e.g., in its chamber) is set in a fixed positionwhen the illumination plane (e.g., light sheet is physically moved upand down) and the detection plane move simultaneously through thespecimen automatically in sync. The detection plane may be moved byautomatically varying the vertical focusing distance of the lens of afocusing unit. In some embodiments, rather than relying on a mechanicalmotor mechanism that may introduce vibrations, the focusing distance ofa focusing unit may be changed electrically (e.g., via an electricallytunable lens that changes focus via electromagnets, piezoelectricelement, current through a solution, etc.) without a use of a motor. Asthe specimen remains in a steady position, vibrations and perturbationissues are alleviated. Incidental specimen mounting and holding becomesmuch easier. Additionally, if a wider field of view of the specimen isdesired, the horizontal sweeping of the “waist” focus of the light sheetas previously discussed may be used in conjunction with the verticalvariable focus. This allows the acquisition of images in the light sheetmode where the image of the portion of the specimen being acquired issynchronized using an electronic synchronization system and isautomatically focused on the thinnest area of the laser beam being sweptvertically and/or horizontally. By synchronizing the detection plane andthe light sheet illumination plane, it is possible to collect thefluorescent emission coming essentially only from the light sheetillumination plane. Hence, it provides the sharpest optical sectioningin the whole frame, greatly reducing shadows occurring within theobserved specimen.

By setting the focal distance of this lens so as to achieve opticalconjugation between the light sheet plane within the specimen and thecamera plane, an image of the selected slice within the specimen can berecorded and/or observed. If required, an electronic system including aprocessor driven by a software program is provided to achieve anautomatic optical coupling between the camera plane and the light sheetplane, thus allowing fast 3D light sheet microscopy. The light sheetscanning capability in the illumination path and associated focusingcapability onto different planes within the observed specimen in thedetection path ensures the optical coupling between the flat illuminatedslice within the specimen and the surface of the digital camera with nomechanical movement of the specimen. Avoidance of the mechanicalmovement of the specimen speeds up collecting light sheet microscopydata, with associated reduction of photo bleaching. Also, this precludesits deformation by the cumulative forces exerted on it by strain relatedwith the interface between the specimen and the water-dipping objectivethrough the matching objective-specimen liquid.

In some embodiments, focal distance of the illumination lens isautomatically adjusted to move and sweep the focus of the lens across arange covering the width of the specimen during image capture toautomatically sweep the thinnest point of the light sheet across thewidth of the specimen, and scanning lines of a rolling shutter of thedetector (e.g., digital camera) are synchronized with the sweepingposition of the waist of the light sheet, allowing a sharper image and alarger field of view without physically moving the specimen within theplane of the light sheet.

By integrating these scanning means, the light sheet system not onlyprovides optical sectioning with optimal spatial resolution and signalto noise ratio, but also delivers unprecedented temporal resolution for3D acquisition, addressing the needs for dynamic imaging of rapidbiophysical processes.

FIG. 6A-FIG. 6C are diagrams illustrating an embodiment of a videooutput focusing unit add-on to an optical microscope. Video outputfocusing unit 602 is coupled to a video output port (e.g., C-Mountinterface) of optical microscope 110 and an optical input of digitalcamera 604 (e.g., via screw-threaded, press fit, friction, locking,bayonet, or any other types of connecting/mounting interfaces). Forexample, focusing unit 602 is included in and/or coupled to detectionunit 112 of FIG. 1B. Digital camera 604 is coupled to focusing unit 602and digital camera 604 captures an image of a specimen obtained via anoptical path of lenses of focusing unit 602 added to the opticaldetection path of microscope 110. Focusing unit 602 has an optical axisthat is substantially parallel to the optical axis of the detectionobjective of the optical microscope for manual or automatic focusingonto the same geometrical plane substantially perpendicular to theoptical axis of the detection objective of the optical microscope, whichis illuminated by the light generated by one or more illuminationsources. Focusing unit 602 includes an arrangement of optical elementswith at least one optical element that is able to dynamically changefocal distance (e.g., tunable lens 610). FIG. 6C shows an internalcutaway view of focusing unit 602. Tunable lens 610 is able to changeits focusing distance electrically (e.g., via electromagnets,piezoelectric element, current through a solution, etc.) without a useof a motor. In other embodiments, focusing distance of focusing unit 602may be changed mechanically.

FIG. 6D-FIG. 6E are diagrams illustrating an embodiment of a detectionobjective output focusing unit add-on to an optical microscope.Detection objective output focusing unit 620 is directly coupled todetection objective 622 and the microscope turret and/or tube lens ofoptical microscope 110 (e.g., via screw-threaded, press fit, friction,locking, bayonet, or any other types of connecting/mounting interfaces).For example, focusing unit 620 functions an intermediary optical elementbetween optical detection objective 622 and a microscope turret ofoptical microscope 110. Digital camera 604 is coupled to focusing unit620 and digital camera 604 captures an image of a specimen obtained viaan optical path of lenses of focusing unit 620 added to the opticaldetection path of microscope 110. Focusing unit 620 has an optical axisthat is substantially parallel to the optical axis of the detectionobjective of the optical microscope for manual or automatic focusingonto the same geometrical plane substantially perpendicular to theoptical axis of the detection objective of the optical microscope, whichis illuminated by the light generated by one or more illuminationsources. Focusing unit 620 includes an arrangement of optical elementswith at least one optical element that is able to dynamically changefocal distance (e.g., tunable lens 622). FIG. 6E shows an external sideview and various internal cutaway views of focusing unit 620. Tunablelens 622 is able to change its focusing distance electrically (e.g., viaelectromagnets, piezoelectric element, current through a solution, etc.)without a use of a motor. In other embodiments, focusing distance offocusing unit 620 may be changed mechanically.

FIG. 7 is a diagram illustrating an embodiment of a specimen holdingchamber assembly. FIG. 7 shows various different view angles of specimenchamber assembly 700. An example of specimen chamber assembly 700 isspecimen chamber and holder assembly 116 in FIG. 1B.

In typical SPIM systems, the specimen is held in a container that isobserved horizontally due to the potential deformability of the specimenembedding medium as a result of the action of gravitational force. Thisis because the optical axis of the detection lens of the microscopecannot extend vertically, as it does in the conventional configurationof the majority of upright or inverted optical microscopes. However, avast majority of the conventional vertical (e.g., upright or inverted)microscopes have detection objective's optical axes extendingvertically. Thus, conventional specimen holding systems for SPIM oftencannot be utilized in these vertical microscopes. Therefore, there is aneed both in providing chambers that are filled with immersion mediumand that can be used in conventional vertical microscopes, comprising anopen top yielding unhindered access to both air and immersionobjectives, a substantially transparent bottom side for viewing thespecimens in transmission mode, and designed to be easily removable fromthe microscope's stand for the microscope to retrieve its original, forexample wide field, functional configuration.

Specimen chamber assembly 700 includes a chamber that is enclosed on thesides and bottom but open on the top (allowing a direct medium immersionfor a detection objective). The bottom of the chamber is substantiallytransparent for observation in transmission mode and coarse specimenpositioning. Two of the sides include substantially transparentcoverslips that allow a light sheet to pass through to illuminate aspecimen placed in the chamber. In some embodiments, the specimen isplaced in the chamber of specimen chamber assembly 700 on a heightadjustable (e.g., by turning a pin/screw/knob) glass support. The glasssupport may be removed from the chamber (or moved away) and a rotarymounting (e.g., T-spike holder) coupled to a cylindrical specimen holderholding a specimen may be placed in the chamber. The rotary mountingcoupled to a cylindrical specimen holder can be laid horizontally in thechamber and the specimen can be rotated about a horizontal axis byrotating a knob and/or via gears that are coupled to a rotatingmechanism (e.g., may be motorized). For example, a specimen is embeddedin a substantially rigid cylindrical transparent embedding compoundmaintained in an immersion liquid and placed in a rotary mount coupledto the chamber. The rotary mount allows a rotational movement of thespecimen using a rotational drive or knob about a substantiallyhorizontal rotational axis and substantially perpendicular to theoptical axis of the detection objective.

In some embodiments, the chamber of assembly 700 is filled with animmersion solution. For example, the chamber that includes a specimen(e.g., either on a glass support or in a cylindrical specimen holder) isfilled with a saline solution, allowing the use of waterdipping/immersion objectives. In some embodiments, to ensure betterresistance against various corrosive agents such as salt water orcleaning agents and ease of cleaning/sonicating/autoclaving,non-transparent parts of chamber assembly 700 are to be made frommedical grade and Polytetrafluoroethylene (e.g., Teflon) parts, suitedto be used together with temperature control equipment for precisetemperature control and equipped with nozzles that allow constant carbondioxide control during experimentation by flowing carbon dioxide on thetop of the chamber. For precise temperature control throughout anexperiment, the baseplate of the chamber is configured for temperaturecontrol. This allows transmission of heating/cooling via contact with aliquid circulation interface for temperature control (e.g., allowingtemperature control from 15° C. to 37° C.). Carbon dioxide control isachieved through nozzles that allow a desired carbon dioxide flow on thetop of the chamber.

FIG. 8 is a diagram illustrating an embodiment of a mold-formed specimenholder over a T-spike rotary mounting. In some embodiments, specimenholder 800 is placed horizontally in the chamber of chamber assembly 700shown in FIG. 7 for observation of the included specimen via SPIMperformed using the system shown in FIG. 1B. Using the conventionalupright or inversed microscope's objective arrangement as a detectionpath of the selective plane light sheet system may require the specimento be rotated about its horizontal axis. In some embodiments, a specimenis embedded in an embedding medium with an increased rigidity sufficientto withstand the actions of gravitational forces when the medium isplaced horizontally. For example, the shown specimen holder allows thespecimen to be embedded within the substantially transparent embeddingmedium in a horizontal direction, perpendicular to the substantiallyvertical orientation of the optical axis of the detection objectivearrangement in upright and inverted microscopes, allowing imaging oftransient events in living biological specimens.

Specimen holder 800 has been formed by molding a substantiallytransparent material in a cylindrical shape over T-spike rotary mounting810. In some embodiments, T-spike rotary mounting 810 includes a medicalgrade Polytetrafluoroethylene material. A specimen is placed inside themolded substantially transparent material and sealed to contain thespecimen within the substantially transparent material even if themolded holder is placed in a horizontal position. The specimen and thespecimen holder may be rotated by rotating the T-spike rotary mounting(e.g., rotation by knob or gear within chamber assembly 700 of FIG. 7placed under a microscope). Prior specimen mounting and specimen holdersolutions for selective plane light sheet microscopy that exist to datehave not been designed to be used with a conventional microscope stand.Unlike the embodiments described herein, prior specimen mounting methodsdo not offer fast, efficient, and reproducible results, neither do theyguarantee stable specimen positioning for the observation and imageacquisition. In some embodiments, an embedding medium is formed withsufficient rigidity to withstand manipulating it around a substantiallyhorizontal direction with repeatable specimen positioning that greatlyalleviates the need for refocusing on the specimen.

FIG. 9 is a flowchart illustrating an embodiment of a process forforming a molded specimen holder. For example, the process of FIG. 9 isutilized to form specimen holder 800 shown in FIG. 8. FIGS. 10A-10Hillustrate an embodiment of various steps of forming a molded specimenholder.

At 902, a T-spike rotary mounting is placed on a vertical stand. In someembodiments, the T-spike rotary mounting is the rotatory mounting shownin FIG. 8. The vertical holder allows the T-spike rotary mounting to bepositioned vertically for the molding process and the mounting isremoved from the vertical stand after the molding process for placementinside a chamber of a chamber assembly (e.g., shown in FIG. 7). Anexample of the placement of the T-spike rotary mounting 1002 on thevertical stand 1004 is illustrated in FIG. 10A.

At 904, a forming mold is placed on the T-spike rotary mounting. Forexample, the forming mold is a hollow cylinder that can be coupled tothe T-spike rotary mounting. The forming mold tightly fits onto therotary mounting to avoid leakage of any liquid material filled in theforming mold. An example of the placement of forming mold 1006 overT-spike rotary mounting 1002 is illustrated in FIG. 10B.

At 906, the forming mold is filled with a substantially transparentmolding material. The molding material may be initially in a liquid orgel state and will solidify over a period of time to become rigid.Examples of the substantially transparent molding material include agar,agarose, gellan gum, or another gelling agent. For example, a Phytagelsolution (e.g., 0.8%) is filled in the forming mold. An example of thefilling of the inside cavity of the forming mold with molding material1008 using a pipette is shown in FIG. 10C.

At 908, a well-shaping mold cap is placed on the forming mold. Thewell-shaping mold cap creates a well in the molding material where aspecimen can be placed. The shown well-shaping mold cap 1010 includes asolid cylindrical extension that is smaller in diameter than thediameter of the interior of the forming mold and when the well-shapingmold cap is capped on the forming mold, the extension of thewell-shaping mold cap is inserted inside the forming mold to occupy andform the space of the specimen well surrounded by the molding material.For example, the cap has a pin extension that will form a pit at the topof the molding material when the molding material solidifies around thepin. The diameter of the cylinder extension/pin is such to host abiological specimen together with the specimen's natural medium. Thewell-shaping mold cap is placed prior to solidification of the moldingmaterial. An example of capping the forming mold with well-shaping moldcap 1010 is shown in FIG. 10D. An example illustration after cappingwith well-shaping mold cap 1010 is shown in FIG. 10E.

At 910, the molding material is allowed to solidify. For example, atroom temperature, a Phytagel molding material solidifies inapproximately five minutes and the amount of time required forsolidification is allowed to pass.

At 912, the shaping molds are removed. For example, the forming mold andthe mold cap are removed. The result is a solidified molding material inthe shape of a cylinder with an open top cylindrical interior well. FIG.1OF illustrates an example of the resulting solidified molding material1012.

At 914, a specimen is inserted in the well of the solidified moldingmaterial along with an appropriate medium. For example, a biologicalspecimen and solution (e.g., solution that is natural, transparent,saline, etc.) are placed inside the well. An example of the placement ofspecimen 1014 suspended in the medium is illustrated in FIG. 10G.

At 916, the well with the specimen is sealed. For example, the well issealed with a substantially transparent material. The sealing materialmay be initially in a liquid or gel consistency that solidifies after aperiod of time. Examples of the sealing material include agar, agarose,gellan gum, or another gelling agent. For example, a low melting agarosegel drop (e.g., ˜1%) is used as the sealing material and is placed onthe opening of the well with the specimen. An example of sealing thewell with sealing material 1016 is illustrated in FIG. 10H.

At 918, the sealing material is allowed to settle and solidify. Forexample, the agarose drop is allowed to settle and solidify forapproximately one minute. By sealing the well, a specimen contained inthe well does not escape the well even if the well is tipped on itsside.

At 920, the specimen holder with the specimen is laid on its sidehorizontally and placed in a chamber assembly for SPIM imaging. Forexample, the specimen holder and the T-spike rotary mounting is placedin the chamber of chamber assembly 700 of FIG. 7. The chamber assemblymay then be placed on the translation stage of the microscope for SPIMimaging.

Although the foregoing embodiments have been described in some detailfor purposes of clarity of understanding, the invention is not limitedto the details provided. There are many alternative ways of implementingthe invention. The disclosed embodiments are illustrative and notrestrictive.

What is claimed is:
 1. A system for illuminating a microscopy specimen,comprising: an illumination source configured to emit a light thattravels along an illumination path to illuminate the microscopy specimenplaced on an optical detection path of an optical microscope; andoptical elements in the illumination path and configured to at least inpart transform the light from the illumination source into a light sheetilluminating the microscopy specimen; wherein the optical elementsinclude an illumination objective and an electronically tunable lensconfigured to vary a focal distance of the electronically tunable lensto dynamically vary a position of a waist of the light sheetilluminating the microscopy specimen; and wherein the optical elementsinclude a deflector configured to vertically move the light sheet toilluminate the microscopy specimen at different horizontal planes,wherein the light sheet extends in the horizontal plane and the verticalmovement of the light sheet corresponds to a movement in a directionperpendicular to the horizontal plane of the light sheet; and whereinthe optical elements include a second deflector configured tohorizontally move a direction with which beam components extendingwithin the light sheet are directed to the microscopy specimen; andwherein the second deflector includes an oscillating mirror thatvibrates about a central rotational axis such that the beam componentsilluminate the microscopy specimen at horizontal light sheet directionsthat alternate many times due to a horizontal light sheet movement atleast in part caused by the second deflector to reduce or eliminate ashadow caused by a substance of the microscopy specimen.
 2. The systemof claim 1, wherein the electronically tunable lens is configured tovary the focal distance of the electronically tunable lens todynamically vary, along an illumination direction, the position of thewaist of the light sheet illuminating the microscopy specimen; andwherein the oscillating mirror of the second deflector is configured tovibrate about the central rotational axis such that the beam componentsilluminate the microscopy specimen at the horizontal light sheetdirections that alternate many times with respect to the illuminationdirection while maintaining a same orientation of the waist of the lightsheet that varies in position along the illumination direction.
 3. Thesystem of claim 1, wherein the deflector includes an oscillating mirrorthat oscillates about a central rotational axis.
 4. The system of claim1, wherein the focal distance of the electronically tunable lens isautomatically swept across a range of focal distance values.
 5. Thesystem of claim 1, wherein the electronically tunable lens is adjustablein a range that includes both positive and negative diopter values. 6.The system of claim 1, wherein the optical elements include a collimatorand one or more diaphragms configured to adjust field or angularaperture stops associated with the light sheet.
 7. The system of claim1, wherein the optical elements include an aspherical optical elementconfigured to at least in part generate a light bundle with an ellipticcross section.
 8. The system of claim 1, wherein the optical detectionpath includes a detector configured to detect a fluorescent lightemitted by the microscopy specimen.
 9. The system of claim 1, whereinthe optical detection path includes a second electronically tunable lensconfigured to automatically vary a focal distance of the secondelectronically tunable lens.
 10. The system of claim 9, wherein thefocal distance of the second electronically tunable lens isautomatically synchronized with automatically varying a verticallocation of the light sheet illuminating the microscopy specimen. 11.The system of claim 1, wherein the optical elements include a group ofoptical relay lenses and an optical objective that directs the lightsheet to the microscopy specimen, and the electronically tunable lens ispositioned in the illumination path between the group of optical relaylenses and the optical objective.
 12. The system of claim 1, wherein theoptical elements include a group of optical relay lenses, and theelectronically tunable lens is positioned in the illumination pathbetween the group of optical relay lenses and the deflector.
 13. Thesystem of claim 1, wherein the optical elements include an asphericaloptical element, and the electronically tunable lens is positioned inthe illumination path between the aspherical optical element and thedeflector.
 14. The system of claim 1, further comprising a motorizedstage configured to move and rotate the microscopy specimen with respectto the light sheet.
 15. The system of claim 1, wherein scanning lines ofa rolling shutter of a detector in the optical detection path aresynchronized with a varying position of the waist of the light sheet.16. The system of claim 1, further comprising a second illuminationsource configured to emit a different light that travels along a secondillumination path to illuminate the microscopy specimen placed on theoptical detection path of the optical microscope.
 17. The system ofclaim 1, wherein the optical microscope is a vertical opticalmicroscope.
 18. A method for illuminating a microscopy specimen,comprising: using an illumination source to emit a light that travelsalong an illumination path to illuminate the microscopy specimen placedon an optical detection path of an optical microscope; and using opticalelements in the illumination path to at least in part transform thelight from the illumination source into a light sheet illuminating themicroscopy specimen; wherein the optical elements include anillumination objective and an electronically tunable lens used to vary afocal distance of the electronically tunable lens to dynamically vary aposition of a waist of the light sheet illuminating the microscopyspecimen; and wherein the optical elements include a deflector used tovertically move the light sheet to illuminate the microscopy specimen atdifferent horizontal planes, wherein the light sheet extends in thehorizontal plane and the vertical movement of the light sheetcorresponds to a movement in a direction perpendicular to the horizontalplane of the light sheet; and wherein the optical elements include asecond deflector used to horizontally move a direction with which beamcomponents extending within the light sheet are directed to themicroscopy specimen; and wherein the second deflector includes anoscillating mirror that vibrates about a central rotational axis suchthat the beam components illuminate the microscopy specimen athorizontal light sheet directions that alternate many times due to ahorizontal light sheet movement at least in part caused by the seconddeflector to reduce or eliminate a shadow caused by a substance of themicroscopy specimen.
 19. The method of claim 18, wherein theelectronically tunable lens is used to vary the focal distance of theelectronically tunable lens to dynamically vary, along an illuminationdirection, the position of the waist of the light sheet illuminating themicroscopy specimen; and wherein the oscillating mirror of the seconddeflector vibrates about the central rotational axis such that the beamcomponents illuminate the microscopy specimen at the horizontal lightsheet directions that alternate many times with respect to theillumination direction while maintaining a same to orientation of thewaist of the light sheet that varies in position along the illuminationdirection.
 20. The method of claim 18, wherein scanning lines of arolling shutter of a detector in the optical detection path aresynchronized with a varying position of the waist of the light sheet.